BIOMEDICINE & PHARMACOTHERAPY
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2022
·
IF 7.419
Qingxia Zuo,Wanqing Xu,Yanbin Wan,Dongyan Feng,Changsheng He,Cailing Lin,Dongchao Huang,Feng Chen,Liya Han,Qi Sun,Dong Chen,Hongli Du,Lizhen Huang
CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.
研究领域:
药理学
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分子生物学
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基因工程
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DOI
PubMed
高影响因子
eLife
·
2020
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IF 7.08
Ce Zhang,Yue Chen,Shijin Sun,Yikai Zhang,Lina Wang,Zhipu Luo,Meng Liu,Liang Dong,Ningzheng Dong,Qingyu Wu
Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin–Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.
研究领域:
细胞生物学
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分子生物学
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肾脏生理学
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DOI
PubMed
高影响因子
JOURNAL OF CONTROLLED RELEASE
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2022
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IF 11.467
Xiaogang An,Renfeng Wang,Erfang Chen,Yang Yang,Bei Fan,Yao Li,Bang Han,Qiong Li,Zhenzhen Liu,Yu Han,Jun Chen,Dingjun Zha
Hearing loss is the most common sensory disorder worldwide and may result from age, drugs, or exposure to excessive noise. Crossing the blood–labyrinth barrier to achieve targeted drug delivery to the inner ear is key to the treatment of hearing loss. We designed a nanoparticle (NP)-based system for targeted drug delivery of forskolin (FSK) to the inner ear, driven by the prestin-targeting peptide LS19 (“ligand–receptor type interaction”). In vivo experiments in developing zebrafish embryos (4–96 h past fertilization) and mice confirmed that LS19-FSK specifically targeted and accumulated in zebrafish lateral line neuromasts and mouse outer hair cells (OHCs). LS19 peptide modification enabled LS19-FSK-NPs to rapidly target OHCs with high specificity. Furthermore, the multifunctional LS19-FSK-NPs were successfully delivered to the OHCs via the round window membrane route and exhibited slow-release properties. The sustained release and intracellular accumulation of FSK inhibited apoptosis of OHCs. Compared with LS19-NPs and FSK-NPs, LS19-FSK-NPs provided significantly stronger protection against noise-induced hearing damage, based on auditory brainstem responses at 4, 8, 16, and 32 kHz. Thus, our specially designed targeted nano-delivery system may serve as a basis for future clinical applications and treatment platforms and has the potential to significantly improve the treatment results of many inner ear diseases .
研究领域:
医学
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药学
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生物技术
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DOI
PubMed
高影响因子
Nanoscience and Nanotechnology Letters
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2018
·Vol. 10
·期 10
·1387-1393
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IF 2.917
Liu Xiaoning,Xu Yuling,Kang Jingjing,Wang Hui,Huang Tao
Mesoporous Silica Nanoparticles (MSNs) loaded with lycorine were previously reported to induce apoptosis of prostate cancer cells with remarkably enhanced efficiency and reduced side effects. In the current study, MSNs-based co-delivery of lycorine and/or the tumor suppressor gene p53
was developed for drug delivery to chronic myelocytic leukemia (CML) cell line K562. We have demonstrated that the simultaneous delivery of lycorine and p53 via MSNs achieved synergistic anti-proliferative and pro-apoptotic effects on K562 cells when compared to MSNs loaded with lycorine only.
The cell viability assay on K562 cells showed that the half-maximal inhibitory concentration (IC50) after 48 h of treatment with MSNs@Lycorine@pcDNA3.1-p53 was only 20% of that for MSNs@Lycorine. Furthermore, the Annexin V-FITC/PI assay for K562 cell apoptosis demonstrated a markedly
higher apoptotic rate in the group treated with MSNs@Lycorine@pcDNA3.1-p53 when compared to that of the MSNs@Lycorine group; this result was consistent with the accumulation of the p53 protein. These findings indicate that MSNs could be an ideal nanovector for simultaneous delivery of drugs
and genes for improved cancer therapy and that an MSN-based lycorine/wt-p53 co-delivery system with efficient endocytosis, sustained drug-releasing property, excellent biocompatibility, and enhanced synergistic proapoptotic effects might be a new promising strategy for the treatment of CML.
研究领域:
药学
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肿瘤学
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纳米医学
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DOI
高影响因子
MICROCHIMICA ACTA
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2018
·Vol. 185
·期 4
·1-7
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IF 5.705
Zhang Kaina,Zeng Ke,Shen Congcong,Tian Shiyu,Yang Minghui
The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L −1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L −1 . This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas.
Fluorescence assay based on hydroxyapatite nanoparticles (HAP) fluorescence quenching was developed for analysis of the activity and inhibition of protein kinase A (PKA).
研究领域:
生物化学
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分子生物学
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药理学
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分析化学
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DOI
PubMed
高影响因子
Journal of Functional Foods
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2024
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IF 3.8
Jinping Wang,Jianmin Wu,Zishen Lin,Ning Ma,Zhaoyue Men,Chao Zhang,Xi Ma,Hongyu Zheng
The invasion of pathogens derived from diet or the environment triggers intestinal inflammation, which is a result of the host intestinal barrier damage and immune dysfunction, such as the decrease of host defense peptides (HDPs) and inhibition of cell proliferation. Herein, the siRNA interference demonstrated that a critical lysozyme, which was mainly induced by dietary supplementations of sodium butyrate and forskolin, improved cell proliferation by inhibiting p38 MAPK and JNK signaling pathways and reinforced the intestinal barrier. Moreover, we established a mouse model of intestinal inflammation with Citrobacter rodentium ( C. rodentium ). It was found that NaBu and FSK stimulated intestinal epithelial proliferation to repair barrier damage, and performed stronger disease resistance to C. rodentium infection on account of the lysozyme upregulation in colon. Therefore, this study revealed that NaBu and FSK prevented or alleviated the development of foodborne infections by promoting lysozyme expression, which may be mediated by the effect of lysozyme on promoting cell proliferation and improving barrier function.
研究领域:
胃肠病学
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免疫学
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分子生物学
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微生物学
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DOI
高影响因子
JOURNAL OF ENDODONTICS
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2019
·
IF 2.833
Jing Zhang,Cheng Fei Zhang,Quan Li Li,Chun Hung Chu
Introduction Stem cells from the apical papilla (SCAPs) possess strong odonto/osteogenic differentiation potential. This study investigated the effect of cyclic adenosine monophosphate (cAMP) on odonto/osteogenic differentiation of SCAPs and the underlining interplay between cAMP and transforming growth factor beta 1 (TGF-β1). Methods SCAPs were stimulated with an activator of cAMP (forskolin) in the presence of either TGF-β1 or a TGF-β1 inhibitor. The amounts of calcium mineral deposition and alkaline phosphatase activity were determined. Quantitative real-time polymerase chain reaction was performed to elucidate cAMP on the TGF-β1–mediated odonto/osteogenic differentiation of SCAPs. The effect of cAMP on the phosphorylation of Smad2/Smad3 and extracellular-regulated kinase (ERK)/P38 induced by TGF-β1 was analyzed by Western blotting . Results Cotreatment with forskolin and a TGF-β1 inhibitor enhanced alkaline phosphatase activity and deposition of calcium minerals in SCAPs. Moreover, the TGF-β1 inhibitor synergized the effect of forskolin on the expression of type I collagen and runt-related transcription factor 2. The results of Western blotting revealed that forskolin attenuated the unregulated expression of the phosphorylation of Smad3 and ERK induced by TGF-β1, and a cAMP inhibitor (H89) antagonized this effect. Conclusions This study showed that cAMP signaling exerts its up-regulating effects on the odonto/osteogenic differentiation of SCAPs by interfering with TGF-β1 signaling via inhibiting Smad3 and ERK phosphorylation.
研究领域:
口腔科学
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细胞生物学
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分子生物学
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DOI
PubMed
高影响因子
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